Genomics research has led to protein therapeutics that are claiming an increasingly large portion of the therapeutic drug market. Protein-based therapies constitute a growing portion of pharmaceutical and biotechnology pipelines. One of the biggest challenges in heterologous protein production on an industrial scale involves the purification of recombinant proteins. Because the protein concentration in the extra-cytoplasmic environment is drastically lower than in the cytoplasm, and often the secreted recombinant protein is the predominant protein in the supernatant, purification of secreted recombinant proteins from extra-cytoplasmic environment proves to be much easier compared to that from the cytoplam. The secreted recombinant protein can be separated from the cells by simple filtration or protein precipitation techniques. This approach of making recombinant proteins secreted has been extremely valuable for the production of numerous proteins, including insulin, growth hormones, various antibodies, and other immunoglobulins, whereby these recombinant proteins are secreted in an unfolded conformation via the universally conserved and essential general secretion (Sec) pathway (Matlack et al., Cell 92:381-390 (1998); Mori et al., Trends in Microbiology 9:494-500 (2001); Pohlschroder et al., Cell 91:563-566 (1997); Fekkes et al., Microbiol. Mol. Biol. Rev. 63:161-173 (1999)).
However, the Sec pathway transports proteins only in an unfolded state, thus it is useless for exporting a significant number of proteins that fold prematurely in the cytoplasm or are unable to fold correctly in the extra-cytoplasmic environment.
Currently, proteins that cannot fold correctly in the extra-cytoplasmic environment must be expressed within the cytoplasm. However, that presents several problems. (i) Proteins folded prior to translocation cannot be secreted into the external environment when fused to a Sec signal sequence. Nevertheless, the Sec signal sequence still targets such folded proteins to the Sec pore, thereby most likely blocking the essential translocation pore, and thus severely affecting host growth. (ii) Purification of heterologously expressed proteins from complex cytoplasmic extracts is extremely challenging and very costly. (iii) Such heterologous proteins are often not stable in the cytoplasmic environment, and tend to aggregate, or be subjected to protein degradation by cytoplasmic proteases. (iv) In some cases cytoplasmically overexpressed proteins form inclusion bodies that are more easily purified, but then resolubilization of such inclusion bodies is often highly challenging and seldom recovers active proteins. Therefore, there is a need for the development of a new method that allows efficient translocation of pre-folded recombinant protein.
Consequently, the focus of the present invention is an alternate secretion mechanism, the twin arginine translocation (Tat) pathway, which secretes proteins in prefolded conformation (Hynds et al., J. Biol. Chem. 273:34868-34874 (1998); Rodrigue et al., J. Biol. Chem. 274:13223-13228 (1999); Thomas et al., Mol. Microbiol. 39:47-53 (2001)). Tat was originally identified in chloroplasts, has since also been found in bacteria and archaea (Santini et al., EMBO J. 17:101-112 (1998); Settles, et al., Science 278:1467-1470 (1997); Voelker et al., EMBO J. 14:3905-3914 (1995); Yen et al., Arch. Microbiol. 177:441-450 (2002).
Analyses of Tat mutants and substrates suggested that the major role of this pathway in Escherichia coli prokaryotes is to translocate redox proteins that integrate their co-factors in the cytoplasm and, therefore, possess some degree of tertiary structure prior to secretion (Berks, 1996; Rodrigue et al., 1999; Weiner et al., Cell 93:93-101 (1998)). However, the recent identification of non-redox Tat substrates (such as, virulence factors from Pseudomonas aeruginosa) indicates a broader role for the pathway in bacterial protein secretion (Ochsner et al., Proc. Natl. Acad. Sci. USA 99:8312-8317 (2001); Voulhoux et al., EMBO J. 20:6735-6741 (2001); Dilks et al., J. Bacteriol. 185(4) (in press 2003)).
At least one copy of a TatA homolog and one copy of a TatC homolog are required for a functional Tat pathway (Bogsch et al., J. Biol. Chem. 273:18003-18006 (1998); Sargent et al., EMBO J. 17:3640-3650 (1998); Yen et al., 2002)). In certain prokaryotes (e.g., E. coli) and many other organisms, multiple copies of TatA have been found and may be involved in the translocation of different substrates, and an additional protein, TatB, has been found to be necessary for Tat-dependent secretion in E. coli (Sargent et al., J. Biol. Chem. 274:36073-36082 (1999)). In B. subtilis, different TatC proteins are expressed under different conditions and seem to be responsible for the translocation of different substrates (Jongbloed et al., J. Biol. Chem. 275:41350-41357 (2000)).
The Tat pathway is distinct from the Sec pathway for, at least, the following reasons: (i) Tat substrates are secreted in a folded conformation (Hynds et al., J. Biol. Chem. 273:34868-34874 (1998); Rodrigue et al., J. Biol. Chem. 274:13223-13228 (1999); Thomas et al., Mol. Microbiol. 39:47-53 (2001)); (ii) Tat signal peptides contain a highly conserved twin arginine motif in their signal sequence (Cristobal et al., EMBO J. 18:2982-2990 (1999); Berks, Mol. Microbiol. 22:393-404 (1996) Berks et al., Mol. Microbiol. 35:260-274 (2000); Chaddock et al., EMBO J. 14:2715-2722 (1995); Niviere et al., J. Gen. Microbiol. 138 (Pt 10):2173-2183 (1992)); (iii) the energy necessary to drive translocation is provided solely by the proton motive force instead of ATP hydrolysis (Cline et al., J. Biol. Chem. 267:2688-2696 (1992); Santini et al., 1998)); and (iv) the Tat pathway is not a universally-conserved secretion mechanism (Wu et al., J. Mol. Microbiol. Biotechnol. 2:179-189 (2000); Yen et al., 2002)).
Because of its unique ability of secreting pre-folded protein substrate, the Tat pathway represents an alternative mechanism for the production of recombinant secreted proteins, particularly for those pre-folded in the cytoplasm of the bacteria. However, in most commonly used bacterial species, such as E. coli, or B. subtilis, the Tat secretion pathway is not as efficient as the Sec pathway, and is not suitable for secreting high level of recombinant proteins under normal physiological growth conditions.
Thus, there is a need to discover or design a more efficient Tat secretion pathway for heterologous protein production and secretion in a biotechnologically amenable organism. To this end, a means is needed for identifying putative Tat substrates. This is necessary to permit investigation of the correlation between the number of components of the Tat system and the number of putative Tat substrates, and to understand how stably and efficiently the folded protein substrate can cross the cytoplasmic membrane.
Microorganisms that naturally utilize the Tat pathway frequently are likely to use the pathway more efficiently. Streptomyces are gram-positive spore-forming soil microorganisms that are well know in the biotechnology for their production of a large variety of secondary metabolites, many of which have antimicrobial, antifungal, and immunosuppressive activities. While particularly noted for antibiotic production, certain strains of Streptomyces have served as hosts for the heterologous production of human proteins with therapeutic value. This is partly due to the availability of well-established plasmid-based expression systems, developed fermentation technology and a low level of endogenous protease activity (Engels and Koller, in Transgenesis—Application of Gene Transfer (Murray, ed.) Wiley & Sons, pp. 32-53 (1992)).
Streptomyces naturally produce many extracellular enzymes, and have the capacity to secrete large amounts of proteins. For example, S. coelicolor has been estimated to secrete as many as 800 proteins into the extra-cytoplasmic environment (Molnar, in Recombinant Microbes for Industrial and Agricultural Applications (Muruoka and Imanaka, eds) Markel Dekker, pp. 81-104 (1994); Gilbert et al., Crit. Rev. Biotechnol. 15:13-39 (1995)). Nevertheless, little is known about the mechanism of the Tat pathway in Streptomyces, let alone as attempt to use the Tat pathway for expressing heterologous proteins.
However, for every therapeutic protein that has been launched, hundreds more are in development. Many of these have been taken off the fast-track due to concerns about ways in which they can be manufactured efficiently, cheaply, and in large quantities. The secretion pathway currently utilized for mammalian cell culture and bacterial expression systems is limited to the production of proteins that fold correctly after translocation to the extra-cytoplasmic environment, but many proteins are incapable of folding correctly under those circumstances due to a lack of appropriate chaperones and other conditions, a fact which has held back the development of countless new treatments. Accordingly, a need has remained to exploit alternative methods to provide for the secretion of folded, active proteins in a highly specific and efficient manner that will be of critical importance to the pharmaceutical industry as it begins to investigate new therapeutic targets identified by the current advances in genomics.